1. Field of the Invention
The invention relates to neuroblastoma cells which were transfected with the recombinant expression plasmid containing aryl hydrocarbon receptor gene. The invention also relates to a method for determining the neurotoxicity of test substance through an aryl hydrocarbon receptor, and a method for obtaining a marker for determining neurotoxicity of the test substance.
2. Description of the Related Art
Neurological disorder such as attention deficit hyperactivity disorder (ADHD) and learning disorder (LD) are major social problems. In the United States of America, about 3 to 5% of children up to age 18 are estimated to suffer from ADHD (Diagnosis and Treatment of Attention Deficit Hyperactivity Disorder, NIH Consensus Statement 1998, Nov. 16-18; 16(2): 1-37); similarly, in Japan, about 3 to 5% of children up to age 18 are also estimated to suffer from ADHD. Accordingly, it is an urgent problem to develop diagnostics and treatments for such neurological dysfunction.
Neurotoxicity of endocrine disruptors has been recently suspected to be a cause of such neurological disorder. The endocrine disruptors exhibit various toxicity such as reproduction toxicity, immunotoxicity and neurotoxicity, and a part of toxicity is known to be expressed through aryl hydrocarbon receptors (AhR) mediated pathway. The aryl hydrocarbon receptor is binding to polycyclic aromatic hydrocarbons (PAHs), and is a transcription factor belonging to bHLH-family. It has been reported that a part of immunotoxicity and reproductive toxicity of the endocrine disrupter is expressed through a pathway mediated by the aryl hydrocarbon receptor (Frank, J. G. et al., Drug Metabolism and Dispositions (1998), 26: 1194-1198). The aryl hydrocarbon receptor is conjectured to be related to neurotoxicity since expression of the aryl hydrocarbon receptor has been detected in brain tissue (Peterson, S. L. et al., J. Comp. Neurol. (2000), 427(3): 428-439). Correlation of expression of neurological functions and developmental formation with gene expression by the aryl hydrocarbon receptor has been also suggested. However, specific genes i.e. marker genes based on activation of transcription by the aryl hydrocarbon receptor in the nervous system hardly ever been identified. Accordingly, it is desired to construct an in vitro culture system suitable for acquiring and analyzing marker genes by analyzing the effect of the aryl hydrocarbon receptor on the neurological function, in order to develop effective diagnostics and treatments for neurological dysfunction.
Appropriate cells should be selected for constructing such an in vitro culture system. First, the cells are required to express the aryl hydrocarbon receptor. It is also desirable to select cells derived from undifferentiated neural cells before differentiation, for example neuroblastoma cells, because the aryl hydrocarbon receptor is considered to act on the nervous developmental process.
Many in vitro culture systems related to the aryl hydrocarbon receptor have been reported. In vitro culture systems using cells for expressing the aryl hydrocarbon receptor, for example Hepa-1 cells derived from mouse hepatic cells and HepG2 cells derived from human hepatic cells (Pollenz, R. S. et al., Mol. Pharmacol. (1999), 56(6): 1127-1137) and MCF-7 cells derived from human breast cancer cells (Wormke M. et al., Mol. Cell Biol. (2003), 23(6): 1843-55), have been constructed, and are used in a wide variety of fields from basic researches to applications such as evaluation of toxicity of chemical substances. Alternatively, it is reported that cells exogenously expressing the aryl hydrocarbon receptor are prepared by transfecting the aryl hydrocarbon receptor gene into cells not expressing the receptor to construct an in vitro culture system. Jarkat cells derived from human leukocytes are included in these cells (Ito, T. et al., J. Biol. Chem. (2004), 279(24): 25204-25210). However, no in vitro systems using cells derived from undifferentiated neural cells have been constructed, and it has been difficult to analyze the effect of the aryl hydrocarbon receptor on the development of the nervous system.
Marker genes and proteins that exhibit changes of expression when the function of the undifferentiated neural cells is expressed have been isolated. Examples of them include tyrosine hydroxylase involved in biosynthesis of dopamine (Bartolini, G. et al., Anticancer Res. (2003), 23(2B): 1495-1499), Cdc42 gene involved in reconstruction of cytoskeleton molecules during formation and outgrowth of neurites (Alleaume, C. et al., Exp. Cell Res. (2004), 299(2): 511-24), and tau gene involved in synapse formation (Knops, J. et al., J. Cell Biol. (1991), 114(24): 725-733). Decision of expression of the neural function of the undifferentiated neural cells is possible by detecting the expression of these molecules. A distinctive change of morphology that neurite outgrowth is observed when the undifferentiated neural cells express their nerve function, or when the cells are differentiated. This means that outgrowth of the neurite serves as a marker for determining the presence of expression of the neural function (Spiegelman B M et al., Cell. (1979), 16(2): 253-263).